The field of the disclosure relates to polymerase chain reactions (PCR) and, particularly, to methods and systems for detecting PCR products during or after the PCR process.
Generally, polymerase chain reaction (PCR) is a process for amplifying nucleic acids and involves the use of two oligonucleotide primers, an agent for polymerization, a target nucleic acid template and successive cycles of denaturation of nucleic acid and annealing and extension of the primers to produce a large number of copies of a particular nucleic acid segment. With this method, segments of single copy genomic DNA can be amplified more than 10 million fold with very high specificity and fidelity. PCR methods are disclosed in U.S. Pat. No. 4,683,202, which is incorporated herein by reference for all relevant and consistent purposes.
PCR was first developed in the 1980s as a method of copying template DNA. The reaction may include DNA polymerase (e.g., Taq-polymerase), building block deoxynucleotide triphosphates (dATP, dTTP, dGTP and dCTP), sequence-specific forward and reverse primer oligonucleotides, a reaction buffer, the template DNA and a thermal cycler. The fundamental PCR reaction begins with a first step (denaturing/melting) at a higher temperature which melts apart the template-paired strands of DNA. This is followed by a second step at a lower temperature (primer annealing) in which the forward and reverse primers attach to the conjugate sequences on the template DNA. The third step (extension/elongation) is at an intermediate temperature in which the DNA polymerase extends the primers by adding paired deoxynucleotides and thus creating the copied deoxynucleic acid strands (cDNA). These three steps are repeated sequentially with a doubling of the product oligonucleotide during each cycle. Typically, the reaction is run for 15 to 40 total cycles.
In practice, the PCR process begins with one long melting step to ensure complete denaturing/melting of the template DNA. Older PCR methods (such as end-point PCR) separate the amplification cycles from the analysis of the amplified products. In other words, a thermal cycler is used to perform the PCR and then the products are analyzed in a separate, second process. This analysis usually involves gel electrophoresis that separates products based on size/molecular weight, or direct oligonucleotide sequencing that determines the actual A, T, C and G base sequences of the product oligonucleotides. The sequence analysis of oligonucleotide products is now more typically performed on complex, automated capillary sequencing systems.
In the late 1990s, a new method of PCR was developed called real-time PCR. This method combined the thermal cycling and detection of the growing oligonucleotide products. These real-time PCR methods employ fluorescent dyes. The commercial real-time PCR systems all integrate a thermal-cycler and an optical fluorescent detection system. These systems typically use a personal computer, but some are stand-alone microprocessor based systems. They also have various numbers of sample wells, including 12-, 24-, 32-, 48-, 96- and 384-well formats.
Product formation and the temperature of product oligonucleotide melting are conventionally determined by thermal analysis of product oligonucleotides via fluorescent based real-time PCR devices. These methods utilize the temperature dependent fluorescence of the sample and require an optical pathway and fluorescent dyes. A need exists for devices and methods for determining oligonucleotide product formation that do not require optical pathways or fluorescent-based analysis.